1. Field of the Invention
The invention relates generally to detecting a biomolecular binding event and more particularly to detecting such events using spectroscopy.
2. Background Information
Many current methods for detecting disease or the risk of developing a disease rely on detection of one or more biomolecular interactions between a target molecule in the biological sample and a detectable probe molecule. The probe molecule is typically detectable because it is bound to a detectable label. For example, infection in a subject caused by an infectious agent, such as a virus, can be detected by detecting binding of a labeled antibody probe to a viral protein. A plethora of bioassays have been developed based on this general concept.
Some more recent methods for detecting disease rely on the detection or determination of a nucleic acid sequence in a test sample. Sequence-selective detection of nucleic acid molecules has become increasingly important as scientists unravel the genetic basis of disease and use this new information to improve medical diagnosis and treatment. Nucleic acid hybridization assays are specific biomolecular binding assays that are commonly used to detect the presence of specific nucleic acid sequences in a sample. For example, an infectious agent can be detected by detecting hybridization of a labeled nucleic acid probe to a nucleic acid of the virus. Alternatively, the method can base disease detection on detection or determination of all or a part of the patient's own nucleic acid sequences. For example, a patient's risk for developing a disease can be determined by detection of a genetic mutation.
Like other methods that detect biomolecular interactions, nucleic acid hybridization assays typically utilize a labeled probe. Traditionally, radioisotopes have been used as labels. More recently, fluorescent, chemiluminescent and bioactive reporter groups have been used. However, the inclusion of labels in an assay often makes it more expensive and complicated, and increases the background signal of the assay.
Hybridization assays can be used not only to detect the presence of a nucleic acid molecule, but determine the sequence of the nucleic acid molecule as well. Traditional approaches for sequence determination utilize the synthesis of labeled nucleic acids that are terminated at one of the four nucleotides. However, these methods are relatively slow and expensive. More recently, methods have been developed that entail synthesizing oligonucleotides on a glass support and effecting hybridization with radioactively or fluorescently-labeled test DNA, and reconstructing nucleotide sequence on the basis of data analysis (E. Southern et al., PCT/GB 89/00460, 1989). A device for carrying out such methods includes a supporting film or glass plate and an array of nucleotides covalently attached to the surface thereof. The array includes a set of oligonucleotides of desired length that are capable of taking part in a hybridization reaction.
The sequencing-by-hybridization method discussed above, although providing a less-expensive method with higher-throughput, has certain disadvantages. For example, it typically requires labeling of sample or probe nucleic acids. As discussed above, this increases the cost and complexity of the method and increases background values, thereby decreasing sensitivity. Furthermore, inclusion and detection of labels lowers the throughput of the assay.
In an attempt to determine nucleic acid sequences information more efficiently, ultraviolet/visible/near-infrared spectroscopy has been used to directly detect hybridization. Although this type of spectroscopy has successfully detected events for smaller molecules (e.g., CO2), it failed to provide the desired level of efficiency and accuracy for biomolecules, which tend to be larger. The frequency shift in the vibration spectrum that is experienced by larger biomolecules (e.g., DNA) upon binding is too small to be accurately and efficiently detected by UV/visible/near-infrared spectroscopy. Furthermore, the UV/visible/near-infrared radiation causes the molecules to fluoresce, creating background noise that interferes with spectrum signals. Further, the method requires multiple gratings of strong dispersion to resolve the small frequency change, making the optical instrument too bulky for convenient use.